The present invention relates to an improvement in or relates to an apparatus and method for obtaining optical density measurements as a means for accurately monitoring a dynamic biomass process system, such as a fermentation, anaerobic or aerobic microorganism process. More specifically, this invention relates to a method for determining the direct optical density of a dynamic process system over the entire operating range by compensation for the primary interference factor.
The measurement of cell density is important in monitoring the program of a fermentation process. There have been numerous methods and accompanying apparatus for measuring cell density. None of the previous methods and apparatus have proved successful and satisfactory before the present invention. Preferably, the use of a sterilizable probe, which can be inserted directly into the fermentor or nutrient media for measurement of the cell density, would be desirable and provide valuable information in monitoring the progress of the process. Normally, product concentration is associated with the cell mass derived from the cell density measurements.
Heretofore, the cell density is measured in discrete intervals by withdrawing samples from the fermentor. This procedure could not produce direct and rapid measurement of the product, but resulted in an estimation of cell mass which was an indication of product concentration at the time the sample was withdrawn from the fermentor. To accomplish satisfactory and accurate process control utilization of a direct measurement system, an on-line measurement system is desirable.
Indirect control measurements are based on various process parameters and are limited by the accuracy of the mathematical model of cell growth, substrate consumption and product formation. Assumption of constant yields, maintenance coefficients, and stoichiometry may not be valid over the entire process range due to depletion of substrates, an accumulation of intermediates which may become metabolized and formation of inibitory products.
In the discrete interval method of cell density measurement at best this is a density approximation technique. From a sterilized sample port, a small volume of culture broth is withdrawn, after assurance the port has been purged. The sample is transported to the laboratory bench and the appropriate dilution of the sample made. Special attention must be paid to accuracy and consistency of the pipetting technique and equipment as between other operators also taking interval samples. Finally, the optical density is measured, multiplied by the dilution factor and the value recorded. During this time, from sample collection to value recording, it is unknown which next appropriate process step is necessary to optimize the overall process.
The discrete interval method of cell density measurement is not conducive to automation. Reliance must be on some indirectly measured parameter: metabolic or physical. Measurements in dynamic systems present various drawbacks. The physical complexity of microbial fermentation cultures is affected by characteristics of the media, the size, shape and type of the organism; changes in the process: pH, temperature, pressure, agitation and the like. Metabolic complexity of growth cultures is influenced by phases of microbial growth in response to the physical environment. This affects cell size, replication duplexes, chain formation, inclusion, body formation and the like. Also for microbes growing in complex nutrient broth, it is not certain which nutrients are utilized at each stage of development in the process. Hence, it is difficult to prepare a model of the growth.
Heretofore, optical sensors would be favorable for measurement of the amount of light that passes through a process fluid. However, the amount of light transmitted through any particular process fluid can be diminished by various factors, such as, suspended solids, dissolved solids and emulsion formation. Suspended solids and emulsions reduce light transmittance by light scattering, as well. In an aerobic system the optical density appears to be influenced by the number of bubbles and their size. Large gas bubbles, such as air bubbles, scatter light just as large particles, but still retain some light transparency. At higher agitation speeds, where smaller bubbles are produced, the scattering of light has a lessened effect, and transparency is increased. Therefore, a biomass system has inherent problems associated with optical sensor measurements.